Mucins, Cystic Fibrosis and PDZ protein interactions

Abstract

Cystic Fibrosis (CF) is caused and characterized by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The dominating feature of the disease is mucus accumulation on mucosal surfaces, but no direct functional connection between mucus and CFTR is known. Mouse models of CF suffer from intestinal mucus accumulation, but do not display the respiratory phenotype that dominates the human disease. Mucins are the major protein components of the mucus layer and they can be subdivided into transmembrane or gel forming. (MUC refers to human, while Muc refers to mouse mucins) We purified guanidinium chloride soluble or insoluble small intestinal mucins from CF mice (Cftrtm1UNC/ Cftrtm1UNC ) and wild type or CF mice that lack expression of the Muc1 transmembrane mucin. We previously suggested that the transmembrane mucin Muc3 is the orthologue of human MUC17 and named the mouse mucin Muc3(17). We demonstrate that the total amount of mucins is increased in CF small intestine. The individual mucins MUC1, Muc2 and Muc3(17) were also increased, with a drastic 1000-fold increase of MUC1. CF-Muc1-/- mice showed the largest amounts of Muc3(17) and in Muc-/- mouse large intestine, mucus thickness was altered. This indicates that Muc1 is able to modulate the biochemical composition and thickness of the mucus layer in the intestine. PDZK1 is a PDZ domain protein known to interact with CFTR. We wanted to identify additional proteins able to bind PDZK1 and study their biological function(s). By performing Glutathione-S Transferase pull down experiments, followed by MALDI-TOF or nanoHPLC mass spectrometry analysis and peptide fingerprinting we could identify MUC17 and the Breakpoint cluster region (Bcr) protein as PDZK1 interacting proteins. By immunofluorescence staining of Muc3(17) on Pdzk1-/- mouse jejunum we detected strong intracellular Muc3(17) staining compared to wild type mice where most of Muc3(17) was located at the apical brush border membrane. Thus, Pdzk1 has a strong influence on the subcellular localization of Muc3(17) in enterocytes, either by acting as an anchor at the apical membrane, or by influencing the apical targeting of the mucin. The facts that PDZK1 is a well known interaction partner of CFTR and that Pdzk1 binds Muc3(17) and stabilizes it at the apical membrane suggest, for the first time a direct connection between CFTR and mucins. The Bcr protein is multifunctional and participates in various signaling pathways. Besides PDZK1 we could identify the vesicle coat protein, Mint-3, as an interaction partner of Bcr. We also discovered two novel motifs in the Bcr amino acid sequence. A clathrin binding box and a consensus sequence for interaction with the AP-2 adapter complex. Both motifs are commonly present in proteins known to participate in clathrin mediated endocytosis. We show that Bcr interacts with both clathrin and á-adaptin and is enriched on clathrin coated vesicles. In BHK-21 cells stably overexpressing Bcr, endocytosis was enhanced and showed a preferred sorting of endocytosed material into lysosomes. It is possible that the interaction with PDZK1 serves as an anchor for Bcr at the plasma membrane where clathrin mediated endocytosis takes place.